Angiogenesis is defined as the growth of new microvessels out of pre-existing capillaries. It may be distinguished from vasculogenesis, which refers to neovascularization during embryonal growth in which also larger vessels are formed and where endothelial precursor cells (EPCs) cells participate. However, there is evidence that EPCs can play a (minor) role in tumor angiogenesis as well. It can also be distinguished from arteriogenesis which mainly involves the maturation and growth of collateral blood vessels (Asahara S. et al 1999, Carmeliet P. 2000, and Helisch A. et al 2003). Angiogenesis is the main mechanism by which new blood vessels are formed during physiological processes like wound healing, inflammation, and the female reproductive cycle. In addition, angiogenesis is involved in various disorders including age-related macular degeneration, rheumatoid arthritis, endometriosis, and cancer (Carmeliet P. et al 2005, and Griffioen A. W. et al 2000). A century ago, it was observed that angiogenesis occurs around tumors and further research led to the hypothesis that tumors produce pro-angiogenesis factors to stimulate neovascularisation (Carmeliet P. et al 2000, Folkman J. et al 1971). The importance of angiogenesis in tumor growth was initially hypothesized in 1971, when Judah Folkman theorized that solid tumors possess limited resources that the many actively proliferating cancer cells fight for. Ever since, tumor angiogenesis research has focused on understanding of and interfering with the processes by which tumor cells promote the growth of new blood vessels (Folkman J. et al 2007, and Ribatti D. et al 2000). The process of tumor angiogenesis is primarily activated when a growing tumor mass surpasses the maximal volume that can be maintained by diffusion of oxygen and nutrients (Carmeliet P. 2000). The hypoxic environment will cause the tumor cells to undergo the angiogenic switch leading to increased production of pro-angiogenesis proteins like the vascular endothelial growth factor (VEGF) (Folkman J. et al 1995, Folkman J. et al 2002, and Hanahan D. et al 1996). These pro-angiogenic proteins activate endothelial cells in nearby vessels. At the same time, an increased activity of different proteolytic enzymes results in degradation of the basal membrane and detachment of cell-cell contacts, which facilitate migration and invasion of EC (endothelial cells) into the surrounding matrix and towards the tumor (Carmeliet P. 2000, and Griffioen A. W. et al 2000). The proteolytic cleavage of the extracellular matrix also allows migration of the activated endothelial cells towards chemotactic signals that originate from the tumor tissue. These signals are sensed by endothelial cells and the subsequent migration and proliferation of endothelial cells results in the formation of vessel-like structures (Adams R. H. et al 2007, and Carmeliet P. 2000). Despite its irregular and disorganized structure, this network is capable of providing the growing tumor mass with all the required metabolites. In addition, the vascular bed provides the tumor cells with the opportunity to enter the circulation and form distant metastases (Folkman J. et al, 2002). While different cell types contribute to neovascularization, the endothelial cell is generally acknowledged to be the central player in the angiogenesis process. In response to different triggers, these cells display a variety of functions, including extracellular matrix remodeling, proliferation, and migration. All these functions require the expression of specific molecules, and proper execution of this complex process relies on endothelial cell flexibility to readily adjust the transcriptome and proteome to comply with the functional demands. Besides the more classical mechanisms, like regulation of gene promoter activity and altered protein turn-over, it has now become evident that cells also use small non-coding RNA molecules to govern gene expression. One class of these RNA molecules, microRNAs (miRNAs), acts as molecular switches that can redirect the expression profile of a cell. Evidence is increasing that these miRNAs fulfil an important role in endothelial gene expression regulation during tumor angiogenesis (Heusschen R, et al 2010).
Many miRNAs show organ-specific expression patterns suggesting cell type-specific functions (Chen C. Z. et al 2004, Poy M. N. et al 2004, and van Rooij E. et al 2007). Consequently, dysregulation of miRNA expression and function may lead to human diseases (Chang T. C. et al 2007). The first large-scale analysis of miRNA expression in endothelial cells (ECs) was carried out in HUVECs (Human Umbilical Venal Endothelial Cells) and identified 15 highly expressed miRNAs with receptors of angiogenic factors (e.g. Flt-1, Nrp-2, Fgf-R, c-Met, and c-kit) as putative mRNA targets, according to prediction algorithms (Polisenol L. et al 2006). Additional studies also profiled the expression of miRNAs in ECs (Kuehbacher A. et al 2007, and Suarez Y. et al 2007). The highly expressed miRNAs that were common in at least 2 of the 3 studies, included miRNA-15b, -16, -20, -21, -23a, -23b, -24, -29a and -b, -31, -99a, -100, -103, -106, -125a, -125b, -126, -130a, -181a, -191, -221, -222, -320, let-7, let-7b, let-7c, and let-7d (Kuehbacher A. et al 2007, Polisenol L. et al 2006, and Suarez Y. et al 2007). However, their specific targets and functions in ECs related to angiogenesis have only been characterized for a few of them.
A study showed that transfection of HUVECs with miRNA-221/222 inhibits tube formation, migration, and wound healing in response to stem cell factor (Polisenol L. et al 2006). This and other studies suggest an antiangiogenic action for these miRNAs and they might be a potential tool to block angiogenesis. However, it is important to note that miR221/222 can also promote cancer cell proliferation through the regulation of p27(Kip1) tumor suppressor (Le Sage C. et al 2007) indicating that the regulation of proliferation by these miRNAs appears cell type specific. Therefore, cell specific targeting with miRNAs is an important area of investigation to be developed.
Other miRNAs expressed in ECs, let-7f and miRNA-27b, have been shown to exert proangiogenic effects, as revealed by the blockade of in vitro angiogenesis with 2′-O-methyl oligonucleotides inhibitors (Kuehbacher A. et al 2007) although their targets in ECs have not yet been characterized.
The best-characterized EC-specific miRNA is miRNA-126 (Fish J. E. et al 2008, Harris T. A. et al 2008, and Wang S. et al 2008). It promotes growth factor (VEGF/FGF Vascular Endothelial Growth Factor/Fibroblast Growth Factor) signaling, angiogenesis, and vascular integrity by inhibiting endogenous repressors of growth factors within ECs (Fish J. E. et al 2008, and Wang S. et al 2008). These findings illustrate that a single miRNA can regulate vascular integrity and angiogenesis, providing a new target for either pro- or antiangiogenic therapies.
A very recent study (Anand S. et al 2010) concludes that miRNA-132 acts as an angiogenic switch by suppressing endothelial p120RasGAP (p120Ras GTPase Activating Protein) expression, leading to Ras activation and the induction of neovascularization, whereas the application of anti miRNA-132 inhibits neovascularization by maintaining vessels in the resting state.
Additionally, several other findings which are not described here provide proof-of-concept for miRNAs as a powerful and highly specific anti-angiogenic therapeutic modality.
There are currently several angiostatic compounds in the market and many are in mid- and late stage clinical testing. Since approximately 5 years, medication based on anti-angiogenesis (e.g. Avastin) has been approved for the clinic. More recently, small molecule RTKI's (Receptor Tyrosine Kinase Inhibitor) are used, e.g. Sunitinib. However, by their nature—targeting essentially tumor driven processes—they evoke clinical resistance. Although there is definitely some prolongation of survival in patient cohorts for some cancer types, the benefit can be considered moderate. In addition to the cancer treatment, three anti-angiogenesis therapies are currently used for the treatment of patients with eye diseases such as wet age-related macular degeneration (AMD): pegaptanib (Macugen, Pfizer), ranibizumab (Lucentis, Novartis), and bevacizumab (Avastin, Roche). Here also, although there is definitely some visual acuity improvement, the benefit is still considered as limited.
Therefore, there is a clear need for better diagnostic markers for neo-angiogenesis, as well as better strategies of therapeutic angiogenesis inhibition.